Apigeninidin-rich Sorghum bicolor (L. Moench) extracts suppress A549 cells proliferation and ameliorate toxicity of aflatoxin B1-mediated liver and kidney derangement in rats

Sorghum bicolor plant has a high abundance of 3-deoxyanthocyanins, flavonoids and other polyphenol compounds that have been shown to offer numerous health benefits. Epidemiological studies have linked increased intake of S. bicolor to reduced risk of certain cancer types, including lung adenocarcinoma. S. bicolor extracts have shown beneficial effects in managing hepatorenal injuries. This study investigated the cytotoxic potential of three apigeninidin-rich extracts of S. bicolor (SBE-05, SBE-06 and SBE-07) against selected cancer cell lines and their ameliorative effect on aflatoxin B1 (AFB1)-mediated hepatorenal derangements in rats. We observed that, among the three potent extracts, SBE-06 more potently and selectively suppressed the growth of lung adenocarcinoma cell line (A549) (IC50 = 6.5 μg/mL). SBE-06 suppressed the expression of STAT3 but increased the expression of caspase 3. In addition, SBE-05, SBE-06 and SBE-07 inhibited oxidative and nitrosative stress, inflammation, and apoptosis and preserved the histoarchitectural networks of the liver and kidney of rats treated with AFB1. These in vitro and in vivo studies indicate the potential of these cheap and readily accessible extracts for cancer therapy and as chemo-preventive agents in preventing aflatoxin-related health issues.


Scheme 2.
Proposed mechanism of SBE-05, SBE-06 and SBE-07 ameliorative effect on AFB 1 -mediated toxicities in the liver and kidney of an experimental rat model. SBE-05, SBE-06 and SBE-07 averted AFB 1induced oxidative and nitrosative stress, inflammation, and apoptosis by attenuating the activity of CYP450 isoforms, NF-kB-mediated generation of pro-inflammatory cytokines, IL-1β as well as altering the Bcl-2/Bax ratio in favour of the action of caspase 9 and caspase 3. Created by https:// app. biore nder. com/. Table 1. List of chemicals, reagents and kits used for the estimation of the oxidative, inflammatory, and apoptotic biomarkers in the liver and kidney of rats.  In vitro cell cytotoxicity assay. A549  Western blot. A549 cells were seeded into a 6-well plate at 1*10 6 /well in DMEM for 24 h. Then, 5 and 10 µg/ mL SBE-06 solutions in DMSO and DMSO were added to the cell culture media such that the final DMSO level is 0.1%. Cells were treated for 24 h, and the medium and cells were scraped from the plate to Eppendorf tubes. The cells in the medium were centrifuged at rpm = 7 for 5 min. Then, the supernatants were discarded, and the pellet was re-suspended with 1 mL of 1 × cold PBS. The cells were centrifuged again at the same setting. The PBS was discarded, and the cells were lysed with RIPA buffer (110μL) (VWR, VWRVN653-100ML) buffer containing phosphatase inhibitor (Fisher Thermo, A32957) and protease inhibitor (Fisher Thermo, A32955). The lysate was vortexed for 30 s, then sonication for 90 s in a water bath. The lysate was then centrifuged at 14,000 rpm for 15 min, and the supernatant was collected and transferred. The total protein concentration was determined using a BCA protein assay kit (BioVision, K813-2500). Based on the results from the BSA assay, the lysates were diluted to make equal protein concentration, and 20-40μg of each lysate was loaded to each well on a TGX MIDI 4-20% gel (Bio-Rad, cat. 5,671,093) and ran at 150 V for 70 min. Subsequently, the gel was transferred to the Turbo PDVF membrane (Bio-Rad, 1,704,273) after blocking with 5% dry milk for 1 h. The membrane was washed three times with TBS-T buffer, and the membrane was incubated overnight with AR (Santa Cruz, sc-7305), p-STAT3/STAT3 (Cell signalling, D3A7/D1B2J), GAPDH (MA116757, Thermo Fisher), and Caspase 3 (Cell signalling, 9662 s/9664 s) antibodies. The membrane was washed with TBS-T for 3 × 5 min on the second day. A secondary antibody (Immunoreagents, part. IR2173) was added, and the membrane was incubated with agitation for one hour. Bands were quantified using the Odyssey CLx Image system.
Animal welfare, sample size calculation and experimental design. The study was conducted in line with the 3R's guidelines, including Replacement, Reduction, and Refinement for the welfare and use of experimental animals 43,44 . Based on these guidelines, we assess the cytotoxic potentials of the apigeninidin-rich fractions in different cancer cell lines. Also, the ameliorative effect of apigeninidin-rich fractions was examined in AFB 1 -challenged rats' hepatorenal system. The sample size for the study was estimated using G*Power Software version 3.1.9.4 45 with an effect size of 0.4 at p < 0.05 for one way analysis of variance (ANOVA) 46 to obtain a total samples size of 125 at 95% power. Out of the 125 animals obtained from our sample size estimation, 48 healthy male Wistar Albino rats, comprising eight cohorts of experimental animals (n = 6) and weighing approximately 164 ± 5 g body weight were purchased from the Experimental Animal Facility of the Faculty of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria. Rats were housed in standard cages placed in an aerated animal house of the Department of Biochemistry, Faculty of Basic Medical Sciences, University of Ibadan, Ibadan, with a 12 h light-dark cycle. Rats were fed with standard rat pellets (purchased from Ladokun Feeds Limited, Ibadan, Nigeria) and water at will and allowed to acclimate for seven days before administration of AFB 1 and SBE. The protocols for the care and use of experimental animals adopted in this study were carried out after the institutional approval was obtained (UI-ACUREC/032-0521-7) and are in conformity with the ratified rules of the University of Ibadan Ethical Committee and the US National Institute of Health. Rats in the eight cohorts of animals were subjected to 28 d successive treatments of AFB 1 and SBE (Fig. 1B). The experimental dose of AFB1 (50 μg/ kg) was adapted from our previous study 14,47 while SBE05, SBE-06, and SBE-07 (5 and 10 mg/kg) were based on data from the cell assay. The gavage volumes taken from specific stock solutions of SBE (5 and 10 mg/kg) and AFB 1 were 0.16, 0.32 and 0.16 mL, respectively prepared by dissolving 150 mg SBE in 30 mL of carboxymethyl cellulose (0.5%) and 5.5 mg AFB 1 in 110 mL of corn oil. Briefly, rats in the control group received 0.32 mL of carboxymethyl cellulose (0.5%), AFB 1 alone received 0. 16  www.nature.com/scientificreports/ Tissue and organ harvest and processing. At the expiration of the 28-day treatment, the rats from all cohorts fasted for 24 h. The whole blood was collected in non-heparinised tubes via retro-orbital venous plexus and allowed to clot. The rats were sacrificed by dislocating their cervical vertebrae after carbon dioxide (CO 2 ) asphyxiation [48][49][50] . The clotted blood samples were centrifuged at 3000 rpm for 10 min. Organs such as the liver and kidney were carefully removed and prepared for biochemical and histological assays after measuring the organ's weight with a USS-DBS16 Analytical Balance (Cleveland, OH, USA). The liver and kidneys of sacrificed rats were further excised into two portions processed for histological investigation and biochemical analyses, respectively. For the biochemical studies, the harvested organs were homogenised −2 g of the liver in 8 mL of homogenizing buffer (in 0.1 M phosphate buffer, pH 7.4) using a glass-Teflon homogeniser. The kidney homogenate was prepared by homogenising the left or right kidney (mean weight: 1.12 g) in 4 mL of the homogenising buffer. The homogenates were centrifuged at 12,000 rpm for 15 min with a cold Eppendorf 5417R centrifuge (Hamburg, Germany). The supernatants (mitochondrial fractions) were collected and frozen in aliquots before biochemical analyses.
Estimation of hepatorenal function parameters. The activities of liver function enzymes-aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP); and kidney functioncreatinine and urea were assayed. Using sera from each cohort of experimental animals and ready to use kits per the manufacturer's directives as reported previously 51 .
Evaluation of oxidative stress, inflammation, and apoptosis in the hepatorenal system. The frozen mitochondrial fractions of the liver and kidney tissues were used to measure the level of oxidative stress, inflammation and apoptosis in the liver and kidney of rats challenged with AFB 1 and remedied with different SBE fractions. The total proteins of the liver and kidney tissues were estimated following an established protocol 52 to determine the exact concentration of any protein of interest in the tissue. The markers of oxidative and nitrosative stress in the liver and kidney of rats were determined following well-documented protocols: Superoxide dismutase (SOD) activity 53,54 ; catalase (CAT) activity 55,56 using H 2 O 2 as a substrate; glutathione S-transferase (GST) activity 57 ; glutathione peroxidase (GPx) activity 58,59 ; reduced glutathione (GSH) level 60,61 . Also, total sulfhydryl (TSH) level 62,63 ; xanthine oxidase (XO) activity 63,64 ; myeloperoxidase (MPO) activity 18,65 ; nitric oxide (NO) level 48,66 were also evaluated accordingly. The level of Malondialdehyde (MDA) and reactive oxygen and nitrogen species (RONS) was determined as previously described 67,68 . Also, biomarkers of inflammation, -IL-1β and IL-10 concentrations-apoptosis -caspase-9 and -3 activities-in the liver and kidney of rats were determined using rat specific enzyme-linked immunosorbent (ELISA) kits following the manufacturer's protocol reported previously 69 . All readings were obtained using a Spectra Max™ 384 Molecular Devices plate reader (San Jose, CA, USA).
Histopathological assessment of the liver and kidney tissues. Before the histopathological examination, each experimental rat's liver and kidney sections were fixed in 10% neutral buffered formalin. The tissues were prepared for histological assessment via a standard paraffin-wax embedded method of 70 as adapted from 71 . Approximately 5 μm thickness of the liver and kidney sections were stained with haematoxylin and eosin, and the slides were coded before being probed with a Carl Zeiss Axio light microscope (Gottingen, Germany). After the examination, representative images taken with a Zeiss Axiocam 512 camera (Gottingen, Germany) attached to the microscope were analysed by a pathologist unaware of the various treatment cohorts from which the slides were prepared.
Statistical analysis. Paired student t-tests were performed to analyse the data generated from this study to determine the significance level in the mean body weight of rats before and after treatment. In addition, a one-way analysis of variance (ANOVA) followed by a post-hoc test (Tukey) was used to identify significant differences across the different cohorts of rats using GraphPad Prism version 8.3.0 for Mac (www. graph pad. com; GraphPad, CA, USA,). The results are expressed as the mean ± SD of replicates, and statistically significant differences were set at a value of p < 0.05.

Ethical approval. All experiments were performed following relevant guidelines and regulations and
adhere to the ARRIVE guidelines (https:// arriv eguid elines. org) to report animal experiments. The protocols for the care and use of experimental animals in this study were approved by the University of Ibadan, Animal Care and Use in Research Ethical Committee with approval number: UI-ACUREC/032-0521-7. The experimental research on S. bicolor complied with all relevant institutional, national, and international guidelines and legislation. The sample of S. bicolor was identified correctly and deposited in the University of Ibadan, Department of Botany and a voucher specimen -Accession number: UIH-23118-was assigned for future reference.

Results
Analysis of fractions from S. bicolor. The isolated solid from each fraction was analysed by LC-MS, monitoring at 420 nm. It was observed that each sample is enriched in a peak with a molecular weight of 255 (retention time = 17.7 min) with variation in the accompanying impurity profiles (Fig. 2i- 3 and Table 2). MDA-MB-231 and Hep-G2 cells are the least responsive to treating the cancer cell lines we investigated. Gratifyingly, SBE-05, SBE-06 and SBE-07 are less toxic to VERO, the normal representative cells (IC 50 range of 55-60 μg/mL; equivalent to 60 mg/kg, assuming a density of 1 g/ mL for the solid). The preceding findings suggest that a dose below 60 mg/kg may be suitable for in vivo evaluation. Intrigued by the enhanced potency of SBE-05, SBE-06 and SBE-07 against A549 cells, we probed further for the effect of SBE-06 on the apoptotic status of A549 cells. We used Western blotting to analyse the effect of SBE-06 on the Signal transducers and activators of transcription 3 (STAT-3) signalling, caspase-3 activation and AR expression 74,75 . We used GAPDH to control for protein loading. While SBE-06 does not affect the levels of  In vivo studies: evaluation of the protective effect of apigeninidin-rich SBE on the hepatorenal system of AFB1-challenged rats. Given that S. bicolor is less susceptible to aflatoxin contamination in the field 37,38 , we next probed further for the effects of SBE-05, SBE-06 and SBE-07 on the hepatorenal system of male Wistar rats challenged with AFB 1 . Specifically, we measured the effect of SBEs on organosomatic indices of rats, including mean body weights, organ weights and relative organ weights of the liver and kidney. Compared to the mean body weight before treatment, animals' final mean body weights increased significantly (p < 0.05) in the control, AFB 1 , and AFB 1 + SBE-07-D 1 experimental cohorts, but non-significantly in other groups. In addition, the mean weight gain of the AFB 1 + SBE-05-D 1 cohort was less than other experimental cohorts (Table 3). Since this measurement is not an index of toxicity, we went further to measure the effects of SBE-05, SBE-06 and SBE-07 on the hepatorenal functional parameters. We observed that SBE-05, SBE-06 and SBE-07 at 5 and 10 mg/kg decreased significantly (p < 0.05) the increase in the activities of AST, ALT, and ALP as well as levels of urea and creatinine, in the sera of rats caused by exposure to AFB 1 . These extracts reduced these hepatorenal indices' activities and levels to quantities comparable to the control cohort's (Fig. 5). Encouraged by this observation, we further investigated the capacity of SBE-05, SBE-06 and SBE-07 to alleviate oxidative stress that results from the exposure of rats AFB 1 . We observed that SBE-05, SBE-06 and SBE-07, at 5 and 10 mg/kg, reversed the AFB 1 -induced alterations in antioxidant-ROS balance in the liver and kidney of rats to levels comparable to that of the control cohorts. Specifically, relative to the AFB 1 -cohort, these extracts increased the activities and levels of SOD, CAT, GST, GPx, GSH, and TSH (p < 0.p05) (Fig. 6) while significantly lowering (p < 0.05) those of XO, MPO, NO, RONS, and LPO (MDA) (Fig. 7). Observing that these extracts could avert AFB 1 -mediated oxidative and nitrosative stress in rats, we further explored their anti-inflammatory and antiapoptotic potentials using the ELISA method. We observed that these extracts decreased significantly (p < 0.05) the concentration of pro-inflammatory cytokine (IL-1β), and activities of caspase-9 and -3, markers of apoptosis (p < 0.05) but  www.nature.com/scientificreports/ significantly lowered the concentration of anti-inflammatory cytokine (IL-10) in the liver and kidney of rats (Fig. 8). Finally, the histological examination of liver and kidney sections revealed that SBE-05, SBE-06 and SBE-07 preserved the histoarchitectural structures of the liver (Fig. 9) and kidney (Fig. 10) similar to that of the control animals. In contrast, the liver and kidney of AFB 1 -challenged rats showed evidence of focal congestion, infiltration of zone 2 by inflammatory cells, mild hydropic/ballooning degeneration of the hepatocytes and moderate Table 3. Body weight gain and relative Liver and Kidney weight of rats following exposure to AFB1 for 28 days. AFB (50 µg/kg); SBE-05-D1, SBE-05-D2, SBE-06-D1, SBE-06-D2, SBE-07-D1 and SBE-07-D2 (10 and 20 mg/Kg respectively) body weight; n = 6. Data are expressed as mean ± SD. ns : p > 0.05 versus Control; ns : p > 0.05 versus AFB1 alone (using ANOVA); *: p < 0.05 versus FBW; ns : p < 0.05 versus FBW (using paired t-test). AFB1 Aflatoxin B1; SBE Sorghum bicolor extract, D1 lower dose; D2 higher dose. www.nature.com/scientificreports/   www.nature.com/scientificreports/ microvesicular steatosis. They disseminated glomerular messangialisation and extensive area of infiltration of the cortex by inflammatory cells, respectively. These observations validate our in vitro cytotoxicity study that the apigeninidin-rich SBE-05, SBE-06 and SBE-07 are relatively less toxic to normal cells and also revealed that they could drive the restoration of cellular and tissue homeostasis in AFB 1 -challenged rats.
To further bridge the knowledge gap on the benefits of S. bicolor in ethnomedicine, we investigated the in vitro anticancer effects of different fractions (SBE05, BSE06, and SBE07) obtained from S. bicolor sheath. Moreover, we probed the abrogative effect of these extracts on AFB 1 -mediated liver and kidney damage in rats. Our LC-MS analysis revealed that these S. bicolor sheath extracts are replete in apigeninidin, a 3-deoxyanthocyanidin widely utilised in the food industry as natural food colourant 86,87 . When screened against selected cell lines-VERO, Hep-G2, A549, MDA-MB-231, MCF-7, LnCaP and DU145-we observed these fractions had enhanced cytotoxicity against A549 in the order SBE-05 < SBE-07 < SBE-06 but were less toxic to the VERO cell. To the best of our knowledge, this is the first report of apigeninidin-rich S. bicolor extracts that elicit selectivity for a lung adenocarcinoma cell line (A549). Previous reports had reported anticancer effects against a wide range of cancer cells, including leukaemia (HL-60) 73 , breast cancer (MCF-7 and MDA-MB 231) 80,88 , cancer cell of the colon (HT-29) and liver (HepG2) 89,90 , malignant cells of colonocytes 91 and hepatoma (Hepa1c1c7) 92 .
Furthermore, our current findings and previous studies agree with epidemiological studies, which indicate that increased intake of S. bicolor is linked to reduced risk of certain types of cancers 93 . To elucidate the mechanism(s) of the anticancer activity of these extracts, we probed the effects of SBE-06, the most potent of the extracts, on the status of selected markers of proliferation and apoptosis in A549 cells. We observed that SBE-06 inhibited the activation of STAT3 via downregulation of phosphorylated STAT3 (p-STAT3) and orchestrated the activation of caspase 3 through induction of the cleavage of cleavage pro-caspase-3. These data show that SBE-06 induced apoptosis and suppressed the proliferation in the A549 cell line by inhibiting the phosphorylation of STAT3 and promoting the activation of pro-caspase-3 ( Fig. 3 & |Scheme 1). Decreased STAT3 expression retards the generation of pro-inflammatory cytokines needed to drive cancer growth, invasion, and metastasis, while increased levels of activated caspase-3 will commit the A549 cells to apoptosis. This observation agrees with previous findings, which revealed that extracts of red sorghum bran rich in 3-deoxyanthocyanidin induce apoptosis through the activation of group II proapoptotic proteins such as Bak and Bax, mediate the release of mitochondrial cytochrome C and apoptosis-inducing factor (PIF) into the cytoplasm and derepressing of caspase-9 and caspase-3 in HL-60 cell line 94   www.nature.com/scientificreports/ Since S. bicolor sheaths contain active principles with antioxidant, anti-inflammatory and chemo-preventive activities, we hypothesise that our extracts are more likely to alleviate the toxic effects of AFB 1 in the rat model. We considered the AFB 1 model of toxicity because it has been reported that AFB 1 exposure orchestrates hepatocellular carcinoma 95 and lung adenocarcinoma in humans 96 , thus making it a public health issue globally 97,98 . To this end, we co-treated adult male Wistar Albino rats with different doses of AFB 1 and SBE-05, SBE-06 and SBE-07. We probed for the effects of this treatment on the levels and activities of crucial hepatic and kidney enzymes in the test animals' sera. The dosages of the extracts that we investigated (5 and 10 mg/kg) are much lower than the concentration of the extracts that induced toxicity to the VERO cells (equivalent to 60 mg/kg). The enzymes we investigated are standard markers of hepatic and renal functions. Specifically, hepatic enzymes ALT and AST are localised in the cytosol while ALP is housed in the mitochondria of hepatocytes and hence are not exuded into the blood. However, these enzymes percolate in hepatocyte damage into the blood, indicating hepatic injury. Urea and creatinine are important biomarkers of renal function and are produced from creatinine phosphate in the muscles and the breakdown of protein, respectively. In a healthy kidney, these by-products are easily removed from the urine by the kidney, but following kidney injuries, they accumulate in the blood, an indication of renal damage 14,63 . We observed that these apigeninidin-rich extracts resisted AFB 1 -mediated derangement in the hepatorenal system of male rats. Specifically, our results showed that these extracts prevented abnormal weight change decreased hepatic and renal dysfunction parameters, including ALT, AST, ALP, GGT, urea and creatinine after 28 d exposure to AFB 1 . This observation is similar to the previous report, which revealed that dye from sorghum bicolor leaf sheath prevented hepatic damage and the induction of oxidative stress in cisplatin-treated rats 99 . To the best of our knowledge, there is no evidence in the pharmacopoeia depicting the protective effect of S. bicolor bioactive compounds in the kidney of rats exposed to toxic agents such as AFB 1 .
Oxidative stress, inflammation and apoptosis are hallmarks of several chronic human diseases and toxic chemicals that orchestrate diseases in humans and animals by disrupting the tight communications between redox, anti/pro-inflammatory and anti/pro-apoptotic signalling different tissues. This study validated earlier observations that AFB 1 triggers RONS generation increase pro-inflammatory cytokines and caspases activity, thereby predisposing rats to hepatorenal damage. These observations agree with the previous findings 14,20 . These effects may have been brought about by AFB 1 capability to drive mRNAs' expression of mRNAs of TNF-α, IL-6, IL-1β, iNOS, COX-2, NF-κB and Keap1a 25,26 ; suppression of Nrf2 and Ho-1 signalling 27,28 and expression of mRNAs of GSH-Px, SOD, CAT, and GST 25,29 . AFB 1 also drive the activation of Bax and Caspase 3 and the expression of mRNAs of FAS, FADD, TRADD and caspase 8 30 . These effects collectively cause oxidative/nitrosative stress, inflammation, and apoptosis in the affected organs. AFB 1 is converted to a toxic metabolite, AFBO by, CYP isoforms (Scheme 2). AFBO can be directly biotransformed by glutathione S-transferase (GST) and other phase-2 enzymes in the liver into AFB 1 -mercapturic acid, collected in the kidney and excreted in the urine. A decrease in the level of GST, especially in the absence of reduced GSH and other total sulfhydryl groups (TSH), will further upturn the AFBO load in the body. AFBO, in this state, drives the expression of XO, whose metabolic action results in the production of reactive oxygen species (ROS), e.g., superoxide anion radical (O 2 .-). O 2 .is immediately converted to hydrogen peroxides (H 2 O 2 ) by superoxide dismutase (SOD). Catalase and glutathione peroxidase convert this toxic by-product into molecular water. This natural remediation process is needed to reduce the liver and kidney levels of AFBO and other AFB 1 metabolites. However, where this remedial process is compromised, the level of these enzymes gradually diminishes with a concomitant elevation in the hydroxyl radical concentrations (OH . ). This process is regarded as the Fenton and Haber-Weiss reactions, and it occurs in the presence of metallic iron (Fe 2+ /Fe 3+ ). The liver and kidneys' myeloperoxidase (MPO) concentrations may increase if H 2 O 2 is not enzymatically neutralised. And may play a role in forming other toxic free radicals, including hypochlorous acid (HOCl). RNS also similarly increases liver and kidney damage. The biochemical processes that lead to the formation of RNS are chiefly contributed by nitric oxide synthases, including inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) 100 , with the evolution of nitric oxide (NO). NO, in the presence of O 2 .-, triggers the generation of peroxyl nitrite (ONOO -). The intermediates -O 2 .-H 2 O 2 , OH . , HOCl, NO, and ONOO --are assayed collectively as indicators of reactive oxygen and nitrogen species (RONS). RONS may interact with cellular biomolecules forming deleterious protein crosslinks, lipid peroxides (malondialdehyde) and DNA adducts in the liver and kidney. Consequently, these adducts will trigger inflammation and apoptosis, harming the hepatorenal system 14,63 . We inferred that these apigeninidin-rich fractions counterbalanced the shifts in redox rheostat as evidenced by the increase in the liver and kidney concentrations of SOD, CAT, GST, GPx, GSH and TSH with a corresponding decrease in XO, MPO, NO, LPO (MDA), and RONS (Scheme 2).
The observed AFB 1 -induced inflammation is ascribed to IL-1β increase and reduced IL-10 in rats' liver and kidneys. Therefore, we infer that the interaction of IL-1β with the IL receptor will result in the activation of MAPK, which in turn activates IKK. IKK is known to mediate the release of NF-kB from NF-kB: IkB complex, which then enters the nucleus and drives the expression of more pro-inflammatory cytokines (Scheme 1). The contribution of IL-1β in the mediation of inflammation and apoptosis has long been established, and current evidence reveals that IL-1β can orchestrate inflammation and apoptosis by stimulating the expression of IL-6, TNF-α, Bax and caspase 3 101 . Our findings show that our apigeninidin-rich extracts resolved AFB 1 -mediated inflammation by lowering the level of IL-1β and increasing IL-10 in the liver and kidney of rats. The anti-inflammatory effect of apigeninidin reported in the current study agrees with previous studies 102 , where apigeninidin was reported to target inflammation through the cyclo-oxygenase-2 (COX-2) and prostaglandin E2 (PGE-2) blockade.
AFB 1 -mediated apoptosis of the liver and kidney is attributed to high caspase 9 and caspase 3-the initiator and executioner of apoptosis, respectively. Based on this observation, it is likely that the increased liver and kidney activities of caspase 9 and caspase 3 may have been brought about by the expression of p53 following alteration in redox balance and unresolved inflammation. The tumour suppressor gene, p53, drives the expression of www.nature.com/scientificreports/ the p53 upregulated modulator of apoptosis (PUMA). This pro-apoptotic protein modulates the Bax/Bcl-2 ratio in favour of the expression of more Bax 103 . Bax then translocates into the mitochondria and causes the evolution of cytochrome C in a process termed mitochondrial permeability transition pore (MPTP). Cytochrome C egress from the mitochondria into the cytoplasm, where it interacts with apoptotic peptidase activating factor 1 (APAF 1) and pro-caspase 9 to form apoptosome. The formation of this complex is needed to activate caspase 9, which then cleaves pro-caspase 3 into caspase 3-the executioner of programmed cell death as observed in the liver and kidney tissues (Scheme 2) as evidenced in our study (Fig. 7). AFB 1 -mediated apoptosis was averted by our apigeninidin-rich extracts as revealed by a decrease in the tissue levels of caspase 9 and caspase 3. These findings corroborate a previous study from our laboratory in which we reported that antioxidant gallic acid prevented liver and kidney damage in AFB 1 challenged rats 14 .
Histoarchitectural evidence is vital for assessing the degree of toxicity impacted on the tissues of animals following exposure to toxic chemicals. It forms the basis for most clinical generalisations after diagnosis 104,105 . It becomes practically impossible to substantiate biochemical and molecular-based claims in clinical and experimental findings without histological evidence. In our current study, AFB 1 disrupted the histoarchitectural frameworks of the liver and kidney tissues. Injury to the hepatorenal tissue is recognised as a hallmark of excessive RONS. These molecules drive the infiltration of monocytes from the blood into AFB 1 -perturbed tissues, where they differentiate to Kupffer cells and intraglomerular mesangial cells 106,107 . These pro-inflammatory cells interact with AFB 1 -mediated danger-associated molecular patterns (DAMPs) through their pattern recognition receptors (PRRs), notably toll-like receptors and drive the activation of NF-κB and interferon regulatory factor (IRF). These transcription factors ingress the nucleus and orchestrate the expression of genes encoding cytokines, notably IL-1β, TNF-α and IFN-α, -β and other soluble mediators. These soluble mediators activate apoptotic or necrotic signalling pathways leading to the death of liver and kidney tissues 108 . Apigeninidin-enriched SBE fractions at the selected doses inhibited severe necrosis and apoptosis of the liver and kidney by increasing the tissue levels of endogenous antioxidants and anti-inflammatory cytokines to inhibit the generation of ROS and RNS and inflammation needed to drive apoptosis and necrosis. Our findings recapitulate the antioxidant and anti-inflammatory effects of apigeninidin-rich SBE and largely agree with previous studies. For instance, bioactive coumarins such as auraptene, marmin, isoauraptene and meranzinhydrate extracted from Citrus grandis peel were reported to inhibit xylene and carrageenan-induced inflammation in vivo 109 while apigenin, luteolin and fisetin depressed inflammation through the downregulation of TNF-α-induced JNK, ERK, p38, CCL2/ MCP-1, and CXCL1/KC activation 110 .

Conclusion
Taken together, our study shows that apigeninidin-rich extracts from S. bicolor selectively inhibited the growth of A549 cells by downregulating the expression of STAT3 and upregulating the activation of caspase-3. Moreover, these extracts averted the induction of oxidative and nitrosative stress, inflammation, and apoptosis in the hepatorenal system of rats. These observations highlight the potential of these cheap and readily accessible extracts for lung cancer therapy and chemo-preventive agents in preventing aflatoxin-related health issues in developing countries. The shortage of improved storage facilities of agricultural products exposes both human and livestock populations to increase aflatoxin contamination risks. Therefore, we recommend that the apigeninidin-rich S. bicolor extracts investigated herein merit extensive preclinical efficacy, toxicology and pharmacokinetic profiling preparatory to their clinical evaluation for managing lung adenocarcinoma and aflatoxicosis in the future.